In pathological diagnosis, first, a sampled tissue is dehydrated and blocked with paraffin to fix the tissue, then the sample is cut into sections having a thickness of 2 to 8 μm followed by removal of the paraffin therefrom, and subsequently the sections are stained to perform microscopic observation. A pathologist performs diagnosis on the basis of morphological information and staining information such as changes in size and shape of cell nuclei and changes in tissue pattern in the microscopic image. The development of image-digitalizing technology has also facilitated wide use of automated pathological diagnosis support equipment that displays information necessary for pathological diagnosis by a pathologist through extraction and measurement of a pathological image input as a digital color image by using a microscope, a digital camera, or any other device in the field of pathological diagnosis.
For example, Patent Document 1 discloses a pathological diagnosis support equipment including a nucleus/cytoplasm distribution-estimating unit for specifying a cell nucleus region and a cytoplasm region from a pathological image; a glandular cavity distribution-extracting unit for specifying a glandular cavity region (region almost not containing a cellular structure) from a pathological image; a cancer site-estimating unit for determining whether or not cancer cells are present; a stage-determining unit for determining the stage of cancer progression; and an image display unit for displaying, for example, a cancer cell distribution map and the stage of progression.
Patent Document 2 discloses a method to detect cancer cells by staining a pathological specimen with two types of dyes selectively and respectively staining the normal site or the cancer site, evaluating the staining concentrations from a spectral image in accordance with Lambert-Beer's law, and determining whether cancer cells are present.
In each method for evaluation, however, the tissue staining is performed by conventional dye staining (e.g., hematoxylin-eosin staining) or dye staining using an enzyme (e.g., DAB staining), and the staining concentration considerably varies depending on environmental conditions such as temperature and time. Accordingly, such pathological diagnosis support equipments cannot maximize its performance in precise quantitative measurement.
Meanwhile, a fluorescent dye having high sensitivity is also used as a labeling reagent in place of the dye described above in study of tissue staining (see Non-Patent Document 1). The present inventors observed a pathological section prepared with an organic fluorescent dye, FITC, under a fluorescence microscope in accordance with the method of identifying/quantitating cells disclosed in Patent Document 3. Unfortunately, the luminescent brightness was too weak to automatically determine a significantly small amount of biomarker on the basis of the light emission level. Hence, the method requires further improvements.